J Young Pharm, 2018; 10(2) Suppl: s97-s100 A multifaceted peer reviewed journal in the field of Pharmacy Original Article www.jyoungpharm.org | www.phcog.net Antimicrobial Activity of Ethyl Acetate Fraction from Stelechocarpus burahol Fruit against Oral Bacteria and Total Flavonoids Content Asni Amin1,3, Maksum Radji2, Abdul Mun’im3*, Anton Rahardjo4, Herman Suryadi5 1Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, University Moeslem of Indonesia, INDONESIA. 2Laboratory of Pharmaceutical Microbiology, Faculty of Pharmacy, Universitas Indonesia, INDONESIA. 3Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Indonesia, Depok, West Java, 16424, INDONESIA. 4Department of Preventive and Public Health Dentistry, Faculty of Dentistry, Indonesia, INDONESIA. 5Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Indonesia, Depok, West Java, 16424, INDONESIA. ABSTRACT Objective: Stelechocarpus burahol is one of the native plants of Indonesia was 125 ug/ml. The total flavonoid content is 0.833 extract mg/g Quercetin that has been used as an oral deodorant. This fruit contains flavonoids, Equivalent. Conclusion: The ethyl acetate fraction of S. burahol fruit contained which flavonoids are known to have antimicrobial activity against oral bacteria. flavonoid that can be used as antimicrobial against oral bacteria. The purpose of this study was to demonstrate the ethyl acetate fraction of S. burahol fruit to inhibit growth of oral bacteria and determine it the Key words: Antimicrobial, Total flavonoid content, Oral bacteria,Stelecho - total flavonoid content. Methods: Antimicrobial activity test was performed carpus burahol, Ethyl acetate fraction. againt oral bacteria (Porphyromonas gingivalis and Fusobacterium nucleatum) Key message: Ethyl acetate fraction of S. burahol fruit with flavonoid content by disc diffusion and microdilution methods. The diffusion assay used can inhibit the growth of oral bacteria. Brucella Blood Agar (BBA) plate with paper disks soaked in the fraction of ethyl acetate S. burahol with various concentration (1,2,4,8,10,20,40, and 60% w/v), the inhibitory zone diameter of each bacteria compared to Correspondence standard catechins. The minimum inhibition concentration value (MIC) was Abdul Mun’im, Laboratory of Pharmacognosy and Phytochemistry, Faculty of using the microdilution method with brucella broth for bacterial growth Pharmacy, Universitas Indonesia, Depok, West Java, 16424, INDONESIA. medium. Total flavonoid content was measured by UV/Visible spectro- Phone: +62 8111184550 photometer with standard quercetin at 440 nm wavelength. Results: The results showed that ethyl acetate fraction could inhibit the growth of Email: [email protected] , [email protected] P. gingivalis and F. nucleatum with concentration of 1% w/v, and the MIC DOI: 10.5530/jyp.2018.2s.19 INTRODUCTION Traditional medicines especially sourced from natural products have halitosis, which it is produced by bacteria P. gingivalis and F. nucleatum. been used to prevent, treat, and maintain human health for a long period The antimicrobial test fromS. burahol extract mouthwash has been of time. Most people living in the developing countries use the medicinal reported and the results show the mouthwash did not exhibit antimicrobial plant for primary healthcare and treat many diseases.1 Nowadays, the activity against P. gingivalis.5-6 uses of traditional medicinal plants for developed as raw drug, new drug P. gingivalis and F. nucleatum are pathogenic bacteria that grow in the compounds, and pharmaceuticals product to health care have steadily oral cavity associated oral disease. Both of these bacteria play an important increased worldwide, include oral healthcare product,2 but research role as the cause of periodontal disease. P. gingivalis are Gram negative and experiment about phytochemicals and pharmacological activity in anaerobic known as black pigmented bacteria that usually cause gingivitis. medicine plant that could be useful as antimicrobials for treatment of Fusobacterium nucleatum belong to the oral microbiota, more commonly infectious diseases, especially antimicrobial against oral bacterial very found in the oral cavity, in gingival pockets as periodontal disease few and limited data.3 One of the medicine plants that have potential for progresses.9-10 oral care is Stelechocarpus burahol (Blume) Hook.f. and Thomson. The Based on phytochemical identification data, it is known that the frac- fruit belonging to family Annonaceae is a traditional herbal medicine in tion of ethyl acetate of S. burahol contains the components of polyphe- Indonesia, which is commonly found in Java Island, especially Central of nols and flavonoids.6 The polyphenols have been reported antimicrobial Java. These fruit was reported has antibacterial and anti-inflammatory activity against oral bacteria to treat dental and periodontal disease. activity.4 The fruits have been empirically used by the princeses of Yo- Polyphenols in foods such as red grape skins and green tea were reported gyakarta Palace for removal oral malodor (halitosis).5-6 Halitosis is oral can inhibit the growth of oral bacteria that cause periodontitis.11-12 diseases in human, commonly derives from the proteolytic activity of Apigenin, galangin, flavone and flavonol glycosides, isoflavones, flavanones, Gram negative anaerobic oral bacteria associated with food metabolism, and chalcones, includes a group of flavonoid compounds have been which the bacteria have a role in degrading the food became amino acids shown to possess potent antimicrobial activity. In addition, some other containing sulfide to produce Volatile Sulphur Compounds (VSCs).7-8 flavonoids such as quercetin, phlorin, naringin, have activity as an oral The S. burahol fruit extracts may inhibit and reduce VSCs causing antibacterial by inhibiting the growth of Gram negative anaerobic bacteria This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. Journal of Young Pharmacists, Vol 10, Issue 2 (Suppl), Apr-Jun, 2018 S97 Amin, et al.: Anti Oral Bacteria of Stelechocarpus burahol associated with periodentitis such as Protovella intermedia, P. gingivalis, and cell growth measured post-incubation. DMSO as the negative control, Actimicitans, F.nucleatum, Solanum morei.13-14 while catechin as the positive control. Minimum inhibitory concentra- Based on the results of in vitro study and still limited information about tion (MIC) values were evaluated by observing the turbidity and the the antimicrobial properties of S. burahol fruit against oral bacteria, we absorbance reading of the suspension post-incubation. The lowest con- want to investigate the antimicrobial activity of the ethyl acetate fraction centrations of burahol fruit fraction without visible growth with clear against oral bacteria and the total flavonoids assay contained in this fruit. suspension were defined as minimum concentrations that inhibited bacterial growth (MIC). To confirm the Minimum Bactericide Concen- MATERIALS AND METHODS tration (MBC), the suspension samples after incubation were transferred Plant material to plate of BBA (conventional plating method) and anaerobically incu- bated for 3x24 h. The lowest concentration with no visible growth was The ripe fruit of S. burahol was collected from smallholder estates in defined as the MBC. The experiment was performed in triplicates. Magelang, Central Java, Indonesia. Harvest time is February-March. The fruit flesh was separated from the pericarp of the fruit and the seeds, the Total Flavonoid Content Assay o fruit flesh was sliced thin and dried at 40 C. Dried fruits powdered and The total flavonoid content (TFC) was conducted according to using the weighed. The sample was ready for the extraction process. aluminum chloride colorimetric method with some modification. The Preparation of S. burahol fraction fraction was dissolved in 10 ml methanol, 0.5 ml of extract solutions were mixed with 2 mL of distilled water, and added 0.2 ml of 10 % AlCl3 Preparation fraction was conducted according partition procedure by solution, 0.2 mL of 1 M sodium acetate, and volume was made up to 5 5-6 Amin et al and Mun’im et al. procedure. Burahol fruit extracted with ml with distilled water, vortex for 2 min and left at room temperature for 15 conventional method by maceration extraction used ethanol solvent. 30 min. The mixture solution incubated for 1 h. The absorbance of the The ethanol extract was dispersed in warm water and ethanol (30.70 ml) reaction mixture was measured at 440 nm with UV/Visible spectropho- and partitioned successively with n-hexane solvent to obtain liquid tometer. The total flavonoid contents were calculated as quercetin from hexane fraction. The residue is re-partitioned with an ethyl acetate solvent. a calibration curve with the solution concentrations of 10 to 60 mg/ml The organic layer is concentrated in a rotary vacuum evaporator under in methanol. The TFC was expressed in mg of quercetin equivalents per reduced pressure at 50°C, to obtain concentrated hexane and ethyl Gram of extract. All the measurements were performed in triplicates.19 acetate fractions. RESULTS The Culture of the Bacterial strains. Antimicrobial Activity Assay Oral bacteria were employed in this